EP3116482B1

EP3116482B1 - Melatonin-based formulations for parenteral administration

Info

Publication number: EP3116482B1
Application number: EP15712818.2A
Authority: EP
Inventors: Annamaria Soliani Raschini; Akif Emre TÜRELI; Eva Marie PRINZ
Original assignee: Chiesi Farmaceutici SpA
Current assignee: Chiesi Farmaceutici SpA
Priority date: 2014-03-13
Filing date: 2015-03-11
Publication date: 2018-08-29
Anticipated expiration: 2035-03-11
Also published as: KR20160132380A; RU2685698C2; CN106102721A; EP3116482A1; HRP20181968T1; ES2687484T3; WO2015135997A1; US9468626B2; KR102239761B1; CN112754997A; AR099751A1; RU2016136327A; RU2016136327A3; US20150258064A1; CA2942111C; CA2942111A1

Description

TECHNICAL FIELD

The present invention relates to a melatonin formulation suitable for parenteral administration.
In particular, the present invention relates to a formulation comprising nanoparticles of melatonin for use in the treatment of neonatal brain injury.

BACKGROUND OF THE INVENTION

Neonates, especially if born prematurely, are very susceptible to free radical oxidative damage. In fact infants at birth are: a) naturally exposed to hyperoxic challenge due to the transition from the hypoxic intrauterine environment to extrauterine life, and this gap is even more significant for neonates that require supplemental oxygen during resuscitation in the delivery room; b) they are more susceptible to infection, especially if born prematurely; c) they have reduced antioxidant defences; d) they possess high levels of free iron that enhances the Fenton reaction causing the production of highly toxic radicals. Oxidative stress likely contributes also to the severity of several neonates diseases as it may affect a variety of organs, often simultaneously, giving rise to different signs according to the organ most damaged. Said diseases include bronchopulmonary dysplasia/chronic lung disease (BDP/CLD), retinopathy of prematurity (ROP), and necrotizing enterocolitis (NEC). Subsequently, it became clear that free radicals are involved in perinatal brain injury as well as in influencing the ductus arteriosus and pulmonary circulation.
In order to counteract free radicals damage many strategies to increase the antioxidant capabilities in term and preterm infants have been proposed and several medications have been experimented with contrasting results.
N-[2-(5-Methoxy-1H-indol-3-yl)ethyl] acetamide, known as melatonin, is an endogenous substance mainly synthesized in the pineal gland from the neurotransmitter serotonin. Melatonin plays a key role in a variety of important physiological functions, including regulation of circadian rhythms, as well as visual, reproductive, cerebrovascular, neuroendocrine, and neuro-immunological actions. Melatonin is a highly effective free-radical scavenger which also enhances the antioxidant potential of the cell by stimulating the synthesis of antioxidant enzymes and by augmenting glutathione levels. Melatonin is also known to counteract cellular energy depletion by preserving mitochondrial homeostasis and protects mitochondrial ATP synthesis by stimulating Complexes I and IV activities. Moreover, melatonin has been shown to attenuate microglial activation and neuroinflammatory responses which are typically associated with hypoxic-ischemic insults. Beside its well documented neuroprotective efficacy, melatonin is an interesting drug, because of its safety profile and its ability to cross all physiological barriers and to reach subcellular compartments.
In light of these properties, during the last decade, melatonin has started to be considered an attractive neuroprotective agent in perinatal asphyxia.
On the other hand, the oral bioavailability of melatonin is low and very variable. Furthermore, melatonin is poorly soluble in water and degrades quickly in water. In the prior art, evidences were reported indicating that melatonin in aqueous solution gradually loses potency at all pH values and is not stable when exposed to light or oxygen. In this respect, it is also well known that some stabilizers and/or preservatives may have the potential to cause toxicological problems, especially in the infant population.
Additionally, the pharmacokinetic profile of melatonin in infants differs from that of adults; therefore dosage of melatonin for term or preterm infants cannot be extrapolated from adult studies. Recently, Robertson N et al (Brain 136(1), 2013, 90-105) have shown that melatonin administered intravenously to newborn piglets increases hypothermic neuroprotection at significantly high doses (5 mg/kg/h). Nevertheless, the formulation utilized in this study is not suitable for administration in human neonates.
WO 2013/068565 A2 discloses melatonin formulations which are administered intravenously for treating neonatal conditions. The formulations are in the form of a powder for reconstitution comprising microparticles of melatonin, a water soluble excipient and a water soluble surfactant. The microparticles have an X90 of less than 100 µm and a VDM of less than 50 µm. It would be highly advantageous to provide a physically and chemically stable, safe formulation suitable for parenteral route for the delivery of high dose of melatonin to neonates for the efficacious treatment of a neonatal disease, preferably neonatal brain injury.
The issue of a safe and effective parenteral delivery of therapeutic doses of melatonin to neonates is solved by the formulation of the present invention.

SUMMARY OF THE INVENTION

In a first aspect, the invention refers to a pharmaceutical formulation comprising a powder to be dispersed in an aqueous vehicle and suitable for parenteral administration to neonates affected by brain injury, said powder consisting of: i) 20 to 75% by weight of nanoparticles consisting of melatonin as active ingredient in admixture with one or more phospholipids having a purity equal to or higher than 99% and selected from the group consisting of phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositol and lecithins, wherein the ratio between melatonin and the phospholipid varies between 90:10 and 20:80 by weight, and ii) 25 to 80% by weight of a mixture of mannitol and trehalose as cryoprotectant agent; wherein at least one of said phospholipid is adsorbed on melatonin surface, and the ratio between mannitol and trehalose is from 6:4 to 4:6 by weight. Advantageously, upon dispersion in the aqueous vehicle, the concentration of melatonin is comprised between 1.0 and 20 mg/ml.
In a second aspect, the invention is directed to a process for preparing the above pharmaceutical formulation comprising the steps of: i) dissolving melatonin and one or more phospholipids in an organic solvent; ii) generating the nanoparticles by controlled precipitation against water as anti-solvent using micro jet reactor technology; iii) adding the cryoprotectant agent; and iv) removing the residual organic solvent and water.
In a third aspect, the invention relates to nanoparticles suitable to be dispersed in an aqueous vehicle consisting of melatonin as active ingredient in admixture with one or more phospholipids having a purity equal to or higher than 99% and selected from the group consisting of phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositol and lecithins, wherein at least one of said phospholipid is adsorbed on melatonin surface, and the ratio between melatonin and the phospholipid varies between 90:10 and 20:80 by weight. In a fourth aspect, the invention is directed to the above melatonin nanoparticles for use in the prophylaxis and/or treatment of a neonatal disease, preferably in the treatment of a hypoxic-ischemic brain injury selected from the group consisting of Hypoxic- Ischemic Encephalopathy (HIE), Perinatal Arterial Stroke (PAS), and Periventricular Leucomalakia (PVL).

DEFINITIONS

With reference to melatonin, the terms "drug", "active ingredient" and "active substance" are used interchangeably.
The term "nanoparticles" means particles having a diameter comprised between 1 and 1000 nanometers in size. Said diameter can be determined according to methods known to the skilled person in the art, for example with Dynamic Light scattering (DLS), and Transmission Electron Microscopy (TEM).
The expression "adsorbed on the surface" means the adhesion of the phospholipid to the surface of the drug. This process creates a film of the phospholipid on the surface. The adsorption of the phospholipid can be determined by FT-IR spectroscopy or by differential scanning calorimetry (DSC), according to procedures known to the skilled person in the art. Typically, as for FT-IR analysis: i) reference spectra of phospholipid and melatonin shall be recorded; ii) to confirm that adsorption occurred, the FT-IR spectrum of the dried nanoparticles should only exhibit the peaks of the phospholipid. As for DSC analysis, the thermal trace of the dried nanoparticles should not show the endothermal melting peak of the drug.
The term "anti-solvent" means a liquid having little or no solvation capacity for the drug.
The term "safe" means a pharmaceutical formulation suitable for injection able of satisfying the injectability criteria for medicinal products, well tolerated by neonates, and devoid of excipients that could be harmful, antigenic or toxic for this patient population.
The expression "water insoluble or poorly water soluble" is used with reference to the solubility in water as defined in the European Pharmacopoeia Ed. 4th, 2003, page 2891. Phospholipids are a class of lipids constituted of glycerol, a phosphate group, a neutral or zwitter-ionic moiety as the characterizing part (choline, serine, inositol etc); The glycerol moiety can be esterified with long chain fatty acids (C₁₄-C₂₂) which in turn can be saturated (e.g. myristic, palmitic and stearic acid), monounsaturated (e.g. oleic acid) or polyunsaturated (e.g. linoleic and arachidonic acid).
Each phospholipid class is a mixture of different species varying for the esterifying fatty acids.
For example, depending on the source, phosphatidylcholines could be constituted of different proportions of: 1,2-dilauryl sn-glycero-3-phosphocholine, generally known as dilauryl-phosphatidylcholine; 1,2-myristoyl sn-glycero-3-phosphocholine, generally known as dimyristoyl-phosphatidylcholine; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, generally known as dipalmitoyl-phosphatidylcholine; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine generally known as palmitoyl-oleoyl-phosphatidylcholine; 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, generally known as palmitoyl-linoleoyl-phosphatidylcholine; 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, generally known as stearoyl-oleoyl-phosphatidylcholine; 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, generally known as stearoyl-linoleoyl-phosphatidylcholine.
The expression "drug loading in the nanoparticles" refers to the ratio of the drug that has been loaded in the nanoparticles to the total content of its dose. It can be determined according to known methods, for example by filtration followed by determination of the residual content of drug in the supernatant. The lesser is the content of the drug in the supernatant, the more efficient is the drug loading. Otherwise, the drug loading could be determined by HPLC assay of the drug upon dissolution of the nanoparticles with ethanol.
The term "extemporaneous preparation" is used to designate all those cases in which the pharmaceutical formulation is not manufactured ready-to-use, rather to be prepared at a time subsequent to that in which the powder is manufactured, usually a time close to the time of administration to the patient.
For a formulation in form of extemporaneous preparation, the expression "chemically stable" refers to a formulation that, upon storage at room temperature (25°C ± 2°C) for at least one day, preferably three days, more preferably one week, shows no drug loss from nanoparticles and no drug degradation.
For a ready-to-use formulation, the expression "chemically stable" refers to a formulation that, upon storage at room temperature (25°C ± 2°C) for at least three days, preferably one week, more preferably one month, even more preferably three months, shows no drug loss from nanoparticles and no drug degradation.
For a formulation in form of extemporaneous preparation, the expression "physically stable" refers to a formulation that, at room temperature (25°C ± 2°C), exhibits substantially no growth in particle size during storage for at least one day, preferably three days, is readily redispersible, and upon redispersion, neither agglomerates nor quick separation from the aqueous vehicle are observed so as to prevent reproducing dosing of the active ingredient.
For a ready-to-use formulation, the expression "physically stable" refers to a formulation that, at room temperature (25°C ± 2°C), exhibits substantially no growth in particle size during storage for at least three days, preferably one week, is readily redispersible, and upon redispersion, neither agglomerates nor quick separation from the aqueous vehicle are observed so as to prevent reproducing dosing of the active ingredient.
The term "therapeutically effective amount" means the amount of the active ingredient, that, when delivered to neonates, provides the desired biological effect.
The term "prophylaxis" refers to the therapeutic use for reducing the occurrence of a disease.
The term "treatment" refers to the therapeutic use for palliative, curing, symptom-allievating, symptom-reducing, disease regression-inducing therapy.

DETAILED DISCLOSURE OF THE INVENTION

Thanks to its antioxidant activity and to its other pharmacological properties, melatonin could be successfully used in the prophylaxis and/or treatment of certain neonatal diseases.
However, the necessity to administer quite high doses makes the development of a liquid formulation difficult.
Therefore, the aim of the present invention is to provide a physically and chemically stable, safe pharmaceutical formulation suitable for parenteral route in neonates, wherein the concentration of melatonin is advantageously comprised between 1.0 and 20 mg/ml.
More advantageously, the concentration shall be comprised between 1.2 and 15 mg/ml, even more advantageously between 3 and 12 mg/ml, preferably between 5 and 10 mg/ml.
In a particular embodiment of the invention, the concentration of melatonin shall be of 5 mg/ml.
Melatonin can be used as a free base or in form of any pharmaceutically acceptable salt and/or solvate thereof.
The pharmaceutical formulation comprises melatonin nanoparticles and a cryoprotectant agent to be resuspended in a aqueous vehicle.
Advantageously, said nanoparticles have a diameter comprised between 20 and 1000 nanometers, more advantageously between 30 and 500 nanometers, even more advantageously between 40 and 350 nanometers, preferably between 60 and 250 nanometers.
In a preferred embodiment, the diameter could be comprised between 100 and 200 nanometers, as said size is appropriate for sterilization by filtration.
The diameter of the nanoparticles has been determined by Dynamic Light scattering (DLS) according to experimental conditions known to the skilled person in the art.
In said nanoparticles, at least one phospholipid is adsorbed on melatonin surface, in such a way as that agglomeration and/or particle growth may be avoided upon resuspension in an aqueous vehicle.
Furthermore, it is well known that a low drug loading may cause homogeneity problems.
Surprisingly, it has been found that by proper selection of the phospholipid, it is possible to achieve a high drug loading in the nanoparticles, advantageously equal to or higher than 65% by weight, preferably higher than 80%, more preferably higher than 90%.
Finally, the stability of the pharmaceutical preparation during its handling and storage may be guaranteed without the need of keeping the nanoparticles in controlled conditions of temperature and/or relative humidity.
Phospholipids are biodegradable, non-toxic, non-antigenic substances which makes them appropriate candidates for parenteral applications.
The phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositol, lecithins, and mixtures thereof. Advantageously the phospholipid has a purity acceptable for parenteral administration to neonates, which is equal to or higher than 99%.
In one embodiment of the invention, if a mixture of phospholipids is intended to be used, lecithins of different sources may be used, for example extracted from soybean oil, sunflower oil or egg yolk. Lecithins having different purities are commercially available from Sigma Aldrich Co, St. Louis, MO, USA or Lipoid AG, Steinhausen, Switzerland or AppliChem GmbH, Darmstadt, Germany.
In other embodiments, single phospholipids of adequate purity and quality for parenteral application may be used, for example, commercially available from LipoidAG. Phosphatidylcholine or hydrogenated phosphatidylcholine are commercially available from Lipoid AG as Lipoid S100 or Phospholipon 80 H, respectively. Phosphatidylcholine of 90% purity is available from Lipoid AG as Lipoid S100. In a preferred embodiment of the invention, a mixture of phosphatidylcholine and lecithin is utilized, preferably in a ratio comprised between 70:30 and 99:1 by weight, more preferably in a ratio comprised between 80:20 and 98:2 by weight. In an aspect not according to the invention, additional agents that could help in stabilizing the nanoparticles (hereinafter stabilization agents) could be added into the formulation to further increase the physical stability of melatonin nanoparticles and modulate the particle size of the nanoparticles. On the other hand, the amount of said stabilizing agent should be suitably controlled to avoid micelle formation.
Advantageously, said stabilization agent could be a tocopherol, preferably D,L alpha-tocopherol also known as vitamin E. In another embodiment, said agent could be deoxycholic acid or a pharmaceutically acceptable salt thereof, preferably the sodium salt. Said stabilization agents could be used alone or in a mixture thereof.
Said agents of purity and quality suitable for parenteral application to neonates are commercially available from Sigma Aldrich Co, St. Louis, MO, USA or Alfa Aesar GmbH, Karlsruhe, Germany. Said stabilization agent could be a C12-C24 saturated or unsaturated fatty acid, preferably C14-C18 saturated or unsaturated fatty acid, more preferably palmitic acid or oleic acid or a pharmaceutically acceptable salt thereof. The stabilization agent could be sodium oleate of purity higher than 99%, commercially available from Lipoid AG.
The cryoprotectant agent is a mixture of mannitol and trehalose in a ratio comprised between 6:4 and 4:6 by weight, more preferably in a 1:1 ratio by weight.
In fact, it has been found that said mixture of cryoprotectant agents significantly improve the redispersibilty of the nanoparticles in form of powder in the aqueous vehicle. Furthermore, the pharmaceutical composition comprising said mixture of cryoprotectant agents turned out to be particularly chemically and physically stable.
Advantageously, the cryoprotectant agent could be present in an amount able of giving rise to a concentration of 5 to 100 mg/ml, preferably from 20 to 80 mg/ml, more preferably between 25 and 50 mg/ml. More advantageously, a cryoprotectant agent of a purity suitable for parenteral administration to neonates shall be utilized, for example available from Sigma Aldrich Co, St. Louis, MO, USA, or BDH Middle East LLC, Qatar.
In a particular embodiment of the invention, melatonin might be present in an amount ranging from 5 to 15% by weight, the phospholipid in an amount comprised between 15 and 60% by weight, and the cryoprotectant agent in an amount comprised between 25 and 80% by weight. The ratio between melatonin and the phospholipid varies between 90:10 and 20:80 by weight.
If in the nanoparticles melatonin is used in combination with a phospholipid and a stabilization agent, the ratio could range between 98:1:1 and 30:40:30 by weight. This embodiment does not fall within the scope of the invention. For example, if melatonin is used in combination with a phosphatidylcholine and deoxycholic acid or a salt thereof, the ratio could range between 5:90:5 and 30:55:15 by weight. More preferably the ratio is 10:80:10 by weight. This embodiment does not fall within the scope of the invention. If melatonin is used in combination with a phosphatidylcholine and oleate sodium salt, the ratio could advantageously range between 10:89.999:0.001 and 30:40:30 by weight. In a particular embodiment, the ratio might be comprised between 10:89.999:0.001 and 15:84.9:0.1 by weight. This embodiment does not fall within the scope of the invention. In this respect, in some exemplary embodiments, the ratio could be: 14.3:85.67:0.03 or 14.3:85.685:0.015 or 14.3:85.693:0.007 or 14.3:85.697:0.003 by weight. These embodiments do not fall within the scope of the invention. In another particular embodiment, the ratio could range from 30:40:30 to 20:60:20 by weight. This embodiment does not fall within the scope of the invention. In this respect, in some exemplary embodiments, the ratio could be 26:47:27 or 23:55:22 by weight. These embodiments do not fall within the scope of the invention. If melatonin is used in combination with lecithin and vitamin E, the ratio could range between 45:50:5 and 96:3:1 by weight, respectively. Exemplary ratios could be 55:43:3 or 76:20:4 or 85:10:5 by weight. These embodiments do not fall within the scope of the invention. The nanoparticles herein disclosed could be stored in a dry solid form and the relative pharmaceutical formulation in form of dispersion prepared extemporaneously before use.
Alternatively, a ready-to-use pharmaceutical formulation could be prepared by dispersing the melatonin nanoparticles and the cryoprotectant agent in a proper aqueous vehicle.
Any pharmaceutically acceptable aqueous vehicle suitable for parenteral administration to neonates could be used, for example water for injection. Otherwise, a saline aqueous solution or a glucose solution could be utilised at a proper concentration that shall be adjusted by the skilled person in the art. In some embodiments, a physiological saline aqueous solution (0.9% w/v sodium chloride) could be preferable.
In other embodiments, a glucose aqueous solution at a concentration of 5% or 10% w/v could advantageously be used.
The pharmaceutical formulation of the invention may comprise other excipients, for instance pH buffers such as acetate, phosphate or citrate buffers, preferably phosphate, and preservatives.
Advantageously the pH of the pharmaceutical formulation is comprised between 4.5 and 8.0, preferably 5.5 and 7.5.
In a particularly preferred embodiment, the pharmaceutical formulation of the invention comprises nanoparticles consisting of 5-15% by weight of melatonin, in admixture with 15-25% by weight of a mixture of phosphatidylcholine and lecithin, and 60-80% by weight of a mixture of mannitol and trehalose as cryoprotectant agent, to be dispersed in a pharmaceutically acceptable aqueous vehicle. Preferably, upon dispersion in said vehicle, the concentration of melatonin is of 5 mg/ml.
Since the pharmaceutical formulation of the invention should be suitable for parenteral administration, its osmoticity is of particular importance. Accordingly, the formulation of the invention shall have an osmolality of less than 600 mOsm/kg advantageously from 180 mOsm/kg to 500 mOsm/kg, more advantageously from 200 to 400 mOsm/kg, preferably from 250 to 350 mOsm/kg.
In a preferred embodiment of the invention, the pharmaceutical formulation of the invention could be administered by intravenous injection or by infusion.
If administered by infusion, the pharmaceutical formulation could be redispersed just before use in a saline or glucose aqueous solution and delivered by a proper infusion pump.
Since it has been reported in the literature than melatonin crosses the placenta, in an alternative embodiment, the pharmaceutical formulation of the invention could be administered antenatal to pregnant women (Miller SL et al J Pineal Res. 2014 Jan 23; Alers NO et al BMJ Open. 2013 Dec 23;3(12)).
Typically, the concentration of the melatonin in the formulation, and hence its dosage will vary with the sex, weight and maturity of the patient, as well as with the severity of the patient's condition. Those of skill in the art will readily be able to determine these factors and to adjust the concentration accordingly.
As an example, the pharmaceutical formulation of the invention could be administered one or more times per day in order to achieve a dosage of 1 to 40 mg/kg/die, advantageously from 5 to 35 mg/kg/die, preferably 30 mg/kg/die.
In one of the preferred embodiments, the formulation is administered by infusion at 5 mg/kg/hour for six hours for a total dosage of 30 mg/kg/die.
The invention further provides a process for preparing the pharmaceutical formulation of the invention, said process comprising the steps of:

i) dissolving melatonin and one or more phospholipids in an organic solvent;
ii) generating the nanoparticles by controlled precipitation against water as anti-solvent using micro jet reactor technology;
iii) adding the cryoprotectant agent; and
iv) removing the residual organic solvent and water.

Depending on the type of phospholipid, suitable organic solvents can be selected from the group including, but not limited to, DMSO, methanol, isopropanol or ethanol, preferably DMSO or ethanol, more preferably ethanol.
However, since residual organic solvents were found to significantly jeopardize the physical stability of the nanoparticles on the invention, they should be removed according to procedures reported in the art.
Preferably they are removed by subjecting the suspension obtained at the end of step iii) to a step of lyophilization according to methods known to the skilled person in the art.
After lyophilisation, the pharmaceutical formulation is harvested to obtain a powder to be reconstituted before use or re-suspended in a proper aqueous vehicle to provide a ready-to-use pharmaceutical formulation.
Details and operative parameters of the micro jet reactor technology are disclosed in US 2011/0294770 . In order to optimize the precipitation step, the person skilled in the art shall properly adjust all the parameters according to its knowledge, in particular the flow rates of the organic solution and water and their mixing ratio.
Optionally, the nanoparticles of the invention and/or the pharmaceutical formulation thereof are sterile.
Sterilization can be achieved according to known methods. For example, the nanoparticles may be sterilized by gamma-irradiation, while the pharmaceutical formulation ready-to-use may be sterilized by filtration or by autoclaving treatments.
The melatonin nanoparticles and pharmaceutical formulations thereof may be used for the prophylaxis and/or treatment of any neonatal disease where there is contribution of an oxidative stress. These diseases include, but are not limited to, bronchopulmonary dysplasia/chronic lung disease (BDP/CLD), retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC) and brain injury due to perinatal asphyxia and hypoxic - ischemic encephalopathy (HIE).
In particular, the melatonin nanoparticles may be used for the prophylaxis and/or treatment of pathologies characterized by cell death, particularly in HIE.
The melatonin nanoparticles may also be used for the prophylaxis and/or treatment of other hypoxic-ischemic neonatal brain injuries encompassing Perinatal Arterial Stroke (PAS), and Periventricular Leucomalakia (PVL).
Nowadays, hypothermia is recognized as an efficacious treatment modality for perinatal asphyxia and HIE. Accordingly, the use of the melatonin nanoparticles in combination with hypothermia may lead to a greater cerebral neuroprotective effect than hypothermia alone, thus improving the immediate and long term clinical outcome.
Since newborns and particularly those delivered prematurely are less protected against oxidation and are highly susceptible to free radical-mediated oxidative damage, the nanoparticles may also be useful to reduce oxidative stress in neonates with sepsis, respiratory distress syndrome or surgical stress.
Moreover, the melatonin nanoparticles may be administered for the prophylaxis and/or treatment of any disease wherein melatonin could be of some benefit, taking into account its known very good safety profile. For example, they may be used as a coadjuvant for many applications including conditions that are typically related to the pediatric age such as:

dyssomnias and difficulties initiating and maintaining sleep. Among these, delayed sleep-phase syndrome (DSPS) and advance sleep-phase syndrome (ASPS);
neurological impairments that affect irregular sleep-wake patterns such as:
- mental or intellectual disabilities, mental retardation, learning disabilities,
- autistic spectrum disorders, Rett syndrome, tuberous sclerosis,
- developmental disabilities and Angelman syndrome;
sleep problems including delayed sleep onset, sleep or bedtime resistance, prolonged tiredness upon waking and daytime sleepiness as well as Attention Deficit Hyperactivity Disorder (ADHD), Smith Magenis Syndrome (SMS) and Sanfilippo Syndrome (SFS).

In a preferred embodiment, as a pharmaceutically acceptable aqueous vehicle, water for injection may be used.
In another embodiment, a physiological saline aqueous solution (0.9% w/v sodium chloride) may be used. The following examples illustrate the preparation of melatonin nanoparticles and of lyophilized formulations comprising the nanoparticles.

Reference Example 1 - Preparation of melatonin nanoparticles in the presence of phosphatidylcholine (purity of 90%)

In order to prepare the nanoparticles, melatonin was dissolved in ethanol in a concentration of 25 or 50 mg/mL in the presence of phosphatidylcholine (Lipoid S 100, Lipoid AG) with concentrations ranging from 5 to 300 mg/mL.
These solutions were precipitated against water using the microjet reactor technology. During the precipitation process, the flow rate of melatonin solution was adjusted to 1-4 ml/min and the flow rate of water was adjusted to 10 mL/min. A gas pressure of 0.1 or 0.2 bar was used to ensure the production of homogenous nanoparticles. The microjet reactor temperature was adjusted to 25-40°C throughout the precipitation process.
Residual organic solvent was removed under vacuum at 30°C.
In order to determine the drug loading, the free melatonin concentration in the aqueous phase nanoparticles were filtered through 0.02 µm syringe filters and centrifuged at 16000 r.p.m. for 90 min.
Melatonin nanoparticles were also characterized in terms of particle size by measuring their diameter through Dynamic Light scattering (DLS).

The results are reported in Table 1.

Table 1

Sample	Conc. Melatonin [mg/mL]	Solvent	Conc phosphatidylcholine [mg/mL]	% Melatonin amount in supernatant	Drug loading in nanoparticles [%]	Particle size [nm]
1	50	Ethanol	16	17.03	82.97	123.4
2	50	Ethanol	18	16.71	83.29	144.9
3	50	Ethanol	20	17.10	82.90	87.75
4	50	Ethanol	22	21.67	78.33	164.7
5	50	Ethanol	24	26.47	73.53	190.1
6	50	Ethanol	26	20.18	79.82	218.5
7	25	Ethanol	8	10.45	89.55	68.83
8	25	Ethanol	8	10.05	89.95	54.37
9	25	Ethanol	10	12.86	87.14	61.23
10	25	Ethanol	10	10.02	89.98	65.25
11	25	Ethanol	12	11.24	88.76	66.05
12	25	Ethanol	12	11.31	88.69	67.05
13	25	Ethanol	18	13.10	86.90	69.06
14	25	Ethanol	18	12.5	87.50	74.67
15	25	Ethanol	22	15.7	84.30	79.63
16	25	Ethanol	22	13.6	86.40	94.09
17	25	Ethanol	26	13.9	86.10	86.5
18	25	Ethanol	26	10.1	89.90	98.44
19	25	Ethanol	5	13.1	86.90	51.29

As it can be appreciated, melatonin nanoparticles with free melatonin content lower than about 15% can be prepared. Some of the samples have a particle size lower than 200 nanometers which is appropriate for filter sterilization.

Reference Example 2 - Preparation of melatonin nanoparticles in the presence of phosphatidylcholine (purity of 90%) and sodium deoxycholate

Melatonin was dissolved in ethanol in a concentration of 25 or 50 mg/mL in the presence of phosphatidylcholine (Lipoid S 100, Lipoid AG) in concentrations ranging from 5 to 200 mg/mL and sodium deoxycholate in concentrations of 15 or 25 mg/mL. During the precipitation, process flow rate of melatonin solution was adjusted to 3-4 ml/min and the flow rate of water was adjusted to 10 mL/min. A gas pressure of 0.2 bar was used to ensure the production of homogenous nanoparticles. Microjet reactor temperature was adjusted to 25-40°C throughout the precipitation process.
The residual organic solvent was evaporated at 30°C under vacuum.
Furthermore, the nanoparticles suspension was made up to volume so as to readjust the active ingredient concentration to approximately 5 mg/ml for all the preparations.
The resulting melatonin nanoparticles were characterized in terms of particle size as reported in Reference Example 1.
Moreover, the total melatonin content in the nanoparticles was determined by HPLC upon their dissolution with ethanol.

The results are reported in Table 2

Table 2

Sample	Conc. Melatonin [mg/mL]	Solvent	Conc. phosphatidylcholine [mg/mL]	Conc. Sodium deoxycholic acid [mg/ml]	Melatonin end conc. [mg/ml]	Particle size [nm]
1	25	Ethanol	75	15	4.91	149.9
2	25	Ethanol	200	25	5.82	152
3	25	Ethanol	125	25	5.54	145.5
4	25	Ethanol	75	15	5.52	164
5	25	Ethanol	200	25	5.86	151.4
6	25	Ethanol	125	25	5.58	123.8

As it can be appreciated, within the experimental error, the concentration of melatonin is consistent with a drug loading higher than 95%.
The particle size is comprised between 100 and 200 nm.

Reference Example 3 - Stability of the melatonin nanoparticles

The sample 5 of Reference Example 2, wherein melatonin, phosphatidylcholine and sodium deoxycholic acid are utilized in a ratio 10:80:10 by weight, was put in a vial and stored at room temperature. After one week of storage, the nanoparticles suspension was characterized for melatonin content and particle size as reported in Reference Example 2.

The results are reported in Table 3.

Table 3

Conc .Melatonin [mg/mL]	Solvent	Conc. phosphatidylcholine [mg/mL]	Conc. Sodium deoxycholic acid	t =0 Melatonin concentration [mg/ml]	t =0 Particle size [nm]	t = 7 dd Melatonin Concentration [mg/ml]	t = 7 dd Particle size [nm]
25	Ethanol	200	25	5.86	151.4	5.97	152.7

As seen above, said formulation proved to be stable for at least one week.

Reference Example 4 - Lyophilised formulation

The samples 1 and 5 of the Reference Example 2 were lyophilised with the addition of mannitol as cryoprotectant agent according to the following program:

Time [h]	00:30	12:00	00:01	06:30	04:30	04:00	01:00	04:00	0.01	30:00
Temperature [°C]	-85	-85	-30	-30	20	20	30	30	30	30
Pressure [mbar]	1.013.25	1.013.25	0.12	0.12	0.12	0.12	0.12	0.12	0.001	0.001

The obtained lyophilized nanoparticles were dispersed in water for injection to obtain varying concentrations of melatonin and mannitol.
The resulting samples were analysed for content and particle size as reported in Reference Example 2.

The results are reported in Table 4.

Table 4

Starting conc melatonin [mg/mL]	Solvent	Starting conc. phosphatidylcholi ne [mg/mL]	Starting conc. Sodium deoxycholic acid [mg/ml]	Mannitol end conc [mg/ml]	Melatoni n end conc [mg/ml]	Particle size [nm]
25.00	Ethanol	75.0	15	7.5	3.21	211.4
25.00	Ethanol			12.5	3.26	199.0
25.00	Ethanol			25.0	3.40	190.8
25.00	Ethanol			50.0	5.07	181.5
25.00	Ethanol	200.1	25	7.5	3.60	214.0
25.00	Ethanol			12.5	4.40	192.6
25.00	Ethanol			25.0	5.03	197.7
25.00	Ethanol			50.0	5.05	175.0

The formulations proved to be well dispersible. In particular, the addition of 25 or 50 mg/mL mannitol gave rise to full dispersity of the nanoparticles after lyophilisation.

Reference Example 5 - Preparation of melatonin nanoparticles in the presence of lecithin and vitamin E

In order to prepare the nanoparticles, melatonin was dissolved in DMSO in a concentration of 150 or 200 mg/mL in the presence of lecithin (Lipoid AG) in a concentration of 0.7 mg/mL and vitamin E in a concentration of 0.5, 1.2 mg/ml.
During the precipitation process flow rates of melatonin solution and water were adjusted in order to have a mixing ratio of 1:1 v/v.
A gas pressure of 0.2 bar was used to ensure the production of homogenous nanoparticles. Microjet reactor temperature was adjusted to 25-40°C throughout the precipitation process.
The residual organic solvent was evaporated at 30°C under vacuum.
Then, the nanoparticles suspension was made up to volume so as to readjust the active ingredient concentration to approximately 5 mg/ml for all the preparations.
The compositions are reported in Table 5. Table 5

Sample Conc. Melatonin [mg/mL] Solvent Conc. Lecithin [mg/mL] Conc. Vitamin E [mg/ml]

1 200 DMSO 0.7 19.8

2 150 DMSO 0.7 4.87

Reference Example 6 - Preparation of melatonin nanoparticles in the presence of phosphatidylcholine (90% of purity) and sodium oleate

Melatonin was dissolved in ethanol in a concentration of 25 mg/mL in the presence of phosphatidylcholine (Lipoid S 100, Lipoid AG) in a concentration of 150 mg/ml and sodium oleate (Lipoid Natriumoleat B, Lipoid AG) in a concentration of 0.05 mg/ml. During the precipitation process, the flow rate of melatonin solution was adjusted to 3-4 ml/min, while the flow rate of water was adjusted to 10 mL/min. A gas pressure of 0.2 bar was used to ensure the production of homogenous nanoparticles. Microjet reactor temperature was adjusted to 25-40°C throughout the precipitation process.
Then, the nanoparticles suspension was made up to volume so as to readjust the active ingredient concentration to approximately 5 mg/ml for all the preparations.
The resulting nanoparticles were characterized in terms of particle size as reported in Reference Example 1.
The results are reported in Table 6. Table 6

Sample Conc. Melatonin [mg/mL] Solvent Conc. phosphatidylcholine [mg/mL] Conc. Na-Oleate [mg/ml] Particle size [nm]

1 25 Ethanol 150 0.05 371.5

2 25 Ethanol 150 0.05 351.2
The samples are then lyophilised upon addition of glycine or mannitol, as reported in Reference Example 4, to achieve a final concentration of 25 mg/ml of the cryoprotectant agent.

Reference Example 7 - Preparation of melatonin nanoparticles in the presence of phosphatidylcholine (90% of purity) and lecithin

Melatonin (50 mg/ml) was dissolved in ethanol in the presence of phosphatidylcholine (Lipoid S 100, Lipoid AG) and soy bean lecithin (Lipoid S PC-3, Lipoid AG) in different ratios. During the precipitation process the flow rate of melatonin solution was adjusted to 2 ml/min and the flow rate of water was adjusted to 10 mL/min. A gas pressure of 0.2 bar was used to ensure the production of homogenous nanoparticles. Microjet reactor temperature was adjusted to 25-40°C throughout the precipitation process.
The samples were lyophilized with the addition of a mixture of mannitol and trehalose in a 1:1 ratio by weight as cryoprotectant agent, according to the conditions of Reference Example 4.
The obtained lyophilized powders were dispersed in water for injection to obtain a concentration of melatonin of about 5 mg/ml for all the preparations.

The compositions of the obtained formulations and the particle size of the melatonin nanoparticles are reported in Table 7.

Table 7

Formulation	Melatonin end conc [mg/mL]	Phosphatidylcholine end conc [mg/mL]	Lecithin end conc [mg/ml]	Mannitol end conc [mg/ml]	Trehalose end conc [mg/ml]	Particle size [nm]
1	4.89	14.67	0.39	25	25	167.6
2	5.27	15.81	1.05	25	25	156.8
3	4.89	16.63	1.47	25	25	186.4

The resulting samples were analyzed for drug loading and particle size as reported in Reference Example 1.
All the formulations proved to be well dispersible with a drug loading higher than 80%.

Reference Example 8 - Stability of the lyophilized pharmaceutical formulation

Samples of lyophilized formulation 1 as prepared in Reference example 7 were stored at 25°C and 40°C in order to evaluate stability of the formulation.
At each time point, lyophilisates were resuspended with water and assessed immediately after resuspension (0h) as well as after 8 hours (8h) and 24 hours (24h) of storage at room temperature (about 25°C).
The chemical stability was checked by HPLC, while the physical stability was visually evaluated. The particle size was determined as reported in Reference Example 1.
The pH was also assessed.

The results are shown in Table 8.

Table 8

		Month 0			Month 1			Month 2			Month 3
ID	[°C]	0h	8h	24h	0h	8h	24h	0h	8h	24h	0h	8h	24h
Assay [mg/ml]	25	4,89	4,82	4,85	4,92	4,84	4,90	4,88	4,85	4,83	4,89	4,83	4,86
Assay [mg/ml]	40				4,94	4,86	4,90	4,93	4,85	4,89	4,95	4,86	4,90
Particle size [nm]	25	167,6	173,8	172,6	166,1	171,3	168,9	176,3	160,8	178,5	165,1	170,8	165,5
	40				164,7	169,8	177,5	165,5	170,5	176,0	175,5	175,7	170,8

pH	25	6,12	6,25	6,25	6,23	6,25	6,35	6,07	6,11	6,12	6,22	6,09	6,12
pH	40				6,03	6,23	6,01	6,07	6,01	6,13	5,91	5,89	5,79

The formulation turned to be chemically and physically stable for at least three months. It also turned out to be stable for at least 24 hours as reconstituted suspension.

Reference Example 9 - Preparation of a pharmaceutical formulation comprising melatonin nanoparticles in the presence of lecithin and vitamin E

In order to prepare the nanoparticles, melatonin was dissolved in ethanol in a concentration of 25 or 50 mg/mL in the presence of lecithin (Lipoid AG) in a concentration of 50 mg/mL, and vitamin E in a concentration of 0.5, 1, 2 or 4 mg/ml.
During the precipitation process flow rates of melatonin solution and water were adjusted in order to have a mixing ratio of 1:2.5 v/v.
A gas pressure of 0.2 bar was used to ensure the production of homogenous nanoparticles. Microjet reactor temperature was adjusted to 25-40°C throughout the precipitation process.
The samples were then lyophilized upon addition of glycine or mannitol, as reported in Reference Example 4, to achieve a final concentration of 50-100 mg/ml of the cryoprotectant agent.

Claims

A pharmaceutical formulation comprising a powder to be dispersed in an aqueous vehicle and suitable for parenteral administration to neonates affected by brain injury, said powder consisting of:
i) 20 to 75% by weight of nanoparticles consisting of melatonin as active ingredient in admixture with one or more phospholipids having a purity equal to or higher than 99% and selected from the group consisting of phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositol and lecithins, wherein the ratio between melatonin and the phospholipid varies between 90:10 and 20:80 by weight, and

ii) 25 to 80% by weight of a mixture of mannitol and trehalose as cryoprotectant agent;
wherein at least one of said phospholipid is adsorbed on melatonin surface, and the ratio between mannitol and trehalose is from 6:4 to 4:6 by weight.
The pharmaceutical formulation according to claim 1, wherein, upon dispersion in the aqueous vehicle, the concentration of melatonin is comprised between 1.0 and 20 mg/ml.
The pharmaceutical formulation according to claims 1 or 2, wherein the phospholipid is phosphatidylcholine or a mixture thereof with lecithin.
The pharmaceutical formulation according to any one of the preceding claims, comprising a further excipient selected from the group consisting of pH buffers, and preservatives.
Nanoparticles suitable to be dispersed in an aqueous vehicle consisting of melatonin as active ingredient in admixture with one or more phospholipids having a purity equal to or higher than 99% and selected from the group consisting of phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositol and lecithins,
wherein at least one of said phospholipid is adsorbed on melatonin surface, and the ratio between melatonin and the phospholipid varies between 90:10 and 20:80 by weight.
A process for preparing the powder according to claim 1 comprising the steps of:
i) dissolving melatonin and one or more phospholipids in an organic solvent;

ii) generating the nanoparticles by controlled precipitation against water as anti-solvent using micro jet reactor technology;

iii) adding the cryoprotectant agent; and

iv) removing the residual organic solvent and water.
The process according to claim 6, wherein the organic solvent and water of step iv) are removed by lyophilisation.
Nanoparticles of claim 5 for use in the prophylaxis and/or treatment of a neonatal disease.
Nanoparticles for use according to claim 8, wherein the neonatal disease is a hypoxic-ischemic brain injury selected from the group consisting of Hypoxic-Ischemic Encephalopathy (HIE), Perinatal Arterial Stroke (PAS), and Periventricular Leucomalakia (PVL).